Aptamer-Based Multispecific Therapeutic Agents

ABSTRACT

Engineered multispecific antigen binding molecules are provided which contain two or more different aptamer moieties joined by a linker. The antigen binding molecules are capable of specifically binding to one or more antigens and bridging different cell types, such as immune cells and cancer cells. The linked aptamers can be used to modulate and enhance immune function.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.62/879,413, filed 26 Jul. 2019; and to U.S. Provisional Application No.62/879,401, filed 26 Jul. 2019; and to PCT Application No.PCT/IB2019/000890, filed 26 Jul. 2019; and to PCT Application No.PCT/US2020/43778, filed 27 Jul. 2020. Each of the aforementionedapplications is hereby incorporated by reference in its entirety.

BACKGROUND

Aptamers are synthetic single strand (ss) DNA or RNA molecules that formspecific secondary and tertiary structures. They can specifically bindto native folded proteins, toxins or other cellular targets with highaffinity and specificity. They are non-immunogenic but like antibodies,aptamers can activate or inhibit receptor functions. Their small size,stability, cost-effective and highly controlled chemical synthesis makeaptamers attractive therapeutic agents. As such, aptamers are regardedas promising synthetic alternatives to monoclonal antibodies for bothdiagnostic and therapeutic purposes

Multispecific aptamers are two or more aptamers linked together anddesigned to specifically bind different epitopes with high affinity andspecificity. The multimeric specificity opens up a wide range ofresearch, diagnostic, and clinical applications, including redirectingcells to another cells type (e.g., T-cell or NK cell to a tumor cell),blocking two different signaling pathways simultaneously, dual targetingof different disease mediators, and delivering payloads to specificcells. In such uses, precise targeting and in some cases the ability toaffect specific cellular function is an important determinant ofsuccessful research, diagnostic and therapeutic uses.

SUMMARY

Provided herein is an engineered antigen binding molecule, comprisingtwo or more different aptamer moieties linked together and capable ofspecifically binding to one or more cancer cell antigens and one or moreimmune effector cell antigens.

An aspect of the invention is a method for linking aptamers of interesttogether. In some embodiments, this can be achieved via click chemistry.In some embodiments the length of the linker, the flexibility ormobility the linker confers to the targeting moieties, as well as thetype of linker can affect immune effector cell function or interferewith the targeting aptamer moieties affecting affinity, specificity, andor conformation. In some embodiments the selection of linker can affectthe pharmacokinetic and pharmacodynamic properties of the multispecificaptamer. In some embodiments the selection of linker can affect activityand safety (e.g., immunogenicity). In some embodiments, the antigenbinding moiety of the multispecific aptamer can recognize with highaffinity and specificity specific antigens.

Another aspect of the invention is a multispecific antigen moleculecontaining two or more linked aptamers having different target bindingspecificities. In some embodiments, the multispecific aptamer can bindand bring within proximity cells expressing the targeted antigens.

In some embodiments, the multispecific aptamer allows for an immuneeffector cell to be redirected to a cancer cell. In turn the binding ofthe engineered multispecific aptamer to the respective targeted epitopesallows for an immune effector cell to become activated and exertunaltered its anti-cancer killing function.

In some embodiments, the antigen binding moiety of the multispecificaptamer can redirect immune effector T-cells expressing CD3, CD8, CD4,or other T-cell specific antigens to other cellular targets of interestsuch as CD19, epithelial cell adhesion molecule, CD20, CD22, CD123,BCMA, B7H3, CEA, PSMA, Her2, CD33, CD38, DLL3, EGF-R, MHC classI-related protein MR1 or Mesothelin.

In some embodiments, the antigen binding moiety of the multispecificaptamer can redirect an immune effector NK cell such as via a CD16A,NKG2D, or other NK-cell specific antigen to other cellular targets ofinterest such as CD30, CD19, Epithelial cell adhesion molecule, CD20,CD22, CD123, BCMA, B7H3, CEA, PSMA, Her2, CD33, CD38, DLL3, EGF-R, MHCclass I-related protein MR1 or Mesothelin.

In some embodiments, the multispecific aptamer can engage conditionalcostimulatory or immune checkpoints by simultaneous targeting of twoimmunomodulating targets, resulting in blockade of an inhibitory target,depletion of suppressive cells, or activation of effector cells (e.g.,involving targets such as PD-1, PD-L1, CTLA04, Lag-3, TIM-3, or OX40)and tumor microenvironment (TME) regulators such as CD47 or VEGF.

In some embodiments, the multispecific aptamer can target one or moretumor associated antigens such as PRAME, NY-ESO-1, MAGE A4, MAGE A3/A6,MAGE A10, AFP.

In some embodiments, the multispecific aptamer can target antigensinvolved in an inflammatory or autoimmune disease, cardiometabolicdisease, respiratory disease, ophthalmic disease, neurologic disease, orinfectious disease.

In some embodiments, the multispecific aptamer is capable of activatingand stimulating immune effector cells to kill cells expressing specifictargeted antigens.

In some embodiments, the multispecific aptamer binds to but does notactivate target cells to which it binds, such as immune effector cells,but merely serves as a bridge between two targets, such as between animmune effector cell and a cancer cell.

In some embodiments, the multispecific aptamer can be a drug productused in the prevention, treatment or amelioration a proliferativedisease, a tumorous disease, an inflammatory disease, an immunologicaldisorder, an autoimmune disease, an infectious disease, viral disease,allergic reactions, parasitic reactions, graft-versus-host diseases orhost-versus-graft diseases in a subject in the need thereof, metabolicdisease, neurologic disease, ophthalmic diseases.

In some embodiments, the multispecific aptamer can be a delivery system(e.g., gene therapy applications).

In some embodiments the multispecific aptamer can be used in diagnosticapplications.

In some embodiments, the multispecific aptamer can be used inpurification systems.

In some embodiments, the multispecific aptamer can be used in cellselection or enrichments applications.

The present technology also can be summarized in the following list offeatures.

1. An aptamer-based multispecific antigen binding molecule comprising 1)two or more target binding aptamer regions having binding specificitiesfor different targets, and 2) one or more linkers connecting the aptamerregions.2. The aptamer-based multispecific antigen binding molecule of feature1, wherein the linker comprises comprises or consists of a linker moietyselected from the group consisting of a covalent bond, a single-strandednucleic acid, a double-stranded nucleic acid, self-assemblingcomplementary oligonucleotides, a peptide, a polypeptide, anoligosaccharide, a polysaccharide, a synthetic polymer, a hydrazone, athioether, an ester, a triazole, a nanoparticle, a micelle, a liposome,a cell, a click chemistry product and combinations thereof.3. The aptamer-based multispecific antigen binding molecule of feature 1or feature 2 that can bind to specific targets on one or more of humancells, immune cells, cancer cells, genetically modified cells, bacteria,or viruses.4. The aptamer-based multispecific antigen binding molecule of any ofthe preceding features that can redirect the binding of one cell typefrom one target cell to another target cell.5. The aptamer-based multispecific antigen binding molecule of any ofthe preceding features that can form a bridge between an immune cell anda cancer cell.6. The aptamer-based multispecific antigen binding molecule of any ofthe preceding features that can stimulate and activate an immune cell.7. The aptamer-based multispecific antigen binding molecule of feature6, wherein the immune cell is a T-cell, NK-cell, or macrophage, and saidbinding leads to destruction of a target cell bound to a target bindingaptamer of the aptamer based multispecific antigen binding molecule.8. The aptamer-based multispecific antigen binding molecule of any ofthe previous features, wherein the molecule possesses a bindingspecificity for an antigen selected from the group consisting of CD3,CD8, CD4, CD19, Epithelial cell adhesion molecule, CD20, CD22, CD123,BCMA, B7H3, CEA, PSMA, Her2, CD33, CD38, DLL3, EGF-R, NKG2D ligands, MHCclass I-related protein MR1, mesothelin, PD-1, PD-L1, CTLA04, Lag-3,TIM-3, OX40, CD47, VEGF, PRAME, NY-ESO-1, MAGE A4, MAGE A3/A6, MAGE A10,and AFP.9. The aptamer-based multispecific antigen binding molecule of feature3, wherein the molecule binds to an immune cell expressing CD3 antigen.10. The aptamer-based multispecific antigen binding molecule of feature1, wherein the molecule binds PSMA antigen on a cancer cell.11. The aptamer-based multispecific antigen binding molecule of feature1 comprising one or more CD3 antigen binding region that can bind to aT-cell and one or more PSMA antigen binding region that can bind to aPSMA expressing cell, wherein the CD3 antigen binding region and thePMSA antigen binding region are connected by one or more linkers.12. Use of the aptamer-based multispecific antigen binding molecule offeature 11 in the treatment of a PSMA expressing cancer includingprostate cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows schematic representations of several embodiments ofmultispecific aptamers of the present technology.

FIG. 2 shows a scheme for a click chemistry reaction to link aptamers.

FIGS. 3A and 3B show binding of anti-PSMA (3A) and anti-CD3 (3B)aptamers to cells that do and do not express the respective antigens.

FIGS. 4A and 4B show agarose gels of monomeric and dimeric (bispecific)aptamers.

FIG. 5 shows the time course (half-life) of an RNA aptamer in serum.

FIGS. 6A and 6B show the binding affinities of bispecific aptamers toPSMA-positive and negative cells.

FIGS. 7A and 7B show the binding affinities of bispecific aptamers toCD3-positive and negative cells.

FIG. 8 shows the cytotoxicity of bispecific aptamers towardsPSMA-positive cells.

DETAILED DESCRIPTION

The linking moiety of an aptamer based multimeric binding molecule canbe simply one or more covalent bonds between individual aptamers or canbe a synthetic or naturally occurring polymer such as a hydrocarbon,polyether, polyamine, polyamide, hydrazone, thioether, ester, triazaole,nucleic acid, peptide, carbohydrate, or lipid. In certain embodiments,the linking moiety is not a peptide. In certain embodiments, the aptamerbased multispecific molecule is devoid of peptides, and is devoid ofpolypeptides and proteins. The linking moiety also can take the form ofa nanoscale structure (such as a polymer, protein, nanoparticle,nanotube, nanocrystal, nanowire, nanoribbon, nanocrystal, micelle, orliposome), or a microscale structure (such as a microbead or a cell), ora larger structure (such as a solid support). Preferably, the linkingmoiety is a biodegradable polymer. The linking moiety can be a polymerthat is linear, branched, cyclic, or a combination of these structures.The linking moiety can also serve as the backbone for a dendrimericstructure, or a hub or star-shaped structure (such as a core structureto which two or more aptamers are bound). For non-covalent association,two or more individual aptamers can be bound via non-covalentinteractions either directly between the aptamers or through interactionwith a linking moiety. The non-covalent interactions can be, forexample, one or more hydrogen bonds, ionic bonds, hydrophobic bonds, vander Waals interactions, or a combination thereof. High affinity bindingpairs, such as streptavidin-biotin, can be used to non-covalently linkaptamers in an aptamer based multimeric binding molecule.

A linker or linking moiety can be any chemical moiety that covalently ornon-covalently joins monomeric aptamer units together. The linker caninclude or consist of, for example, oligonucleotides, polynucleotides,peptides, polypeptides, or carbohydrates. The linker can include orconsist of a cell receptor, a ligand, or a lipid. The linker can includeor consist of a hydrocarbon chain or polymer such as a substituted orunsubstituted alkyl chain or ring structure, a polyethylene glycolpolymer, or a modified or unmodified oligonucleotide or polynucleotide.The linker can be a single covalent bond, or can include one or moreionic bonds, hydrogen bonds, hydrophobic bonds, or van der Waalsinteractions. The linker can include a disulfide-bridge, a heparin orheparan sulfate-derived oligosaccharide (a glycosoaminoglycan), achemical cross-linker, hydrazone, thioether, ester, or triazole. Thelinker can be cleavable by an enzyme, allowing for release of individualapttamers and/or termination of a target-target interaction by theinteraction by the aptamer based multispecific molecule. The linker canhave a net positive, negative, or neutral charge. The linker can be asflexible or as rigid as desired to ensure preservation of the functionalproperties of the individual monomeric aptamer units in a multimericconstruct and to promote binding to the first target and the secondtarget, or to promote their interaction. The linker can include aflexible portion, such as a polymer of 5-20 glycine and/or serineresidues. The linker can also contain a rigid, defined structure, suchas a polymer of glutamate, alanine, lysine, and/or leucine. The linkercan include a hinge portion or a spacer portion. The linker can includea substituted or unsubstituted C2-C50 chain or ring structure, apolyethylene glycol polymer (e.g., hexaethyleneglycol), or a modified orunmodified oligonucleotide or polynucleotide. The linker can include aheparin or heparan sulfate-derived oligosaccharide (aglycosoaminoglycan), a chemical cross-linker, peptide, polypeptide,hydrazone, thioether, or ester.

A C2-C50 linker can include a backbone of 2 to 50 carbon atoms(saturated or unsaturated, straight chain, branched, or cyclic), 0 to 10aryl groups, 0 to 10 heteroaryl groups, and 0 to 10 heterocyclic groups,optionally containing an ether linkage, (e.g., one or more alkyleneglycol units, including but not limited to one or more ethylene glycolunits —O—(CH2CH2O)—; one or more 1,3-propane diol units; an amine, anamide; or a thioether. Each backbone carbon atom can be independentlyunsubstituted (i.e., comprising only —H substituents) or can besubstituted with one or more groups selected from C1 to C3 alkyl, —OH,—NH2, —SH, —O—(C1 to C6 alkyl), —S—(C1 to C6 alkyl), halogen, —OC(O)(C1to C6 alkyl), and —NH—(C1 to C6 alkyl). In some embodiments, the linkeris a C2-C20 linker, a C2-C10 linker, a C2-C8 linker, a C2-C6 linker, aC2-C5 linker, a C2-C4 linker, or a C3 linker, wherein each carbon may beindependently substituted as described above.

In certain embodiments, there is non-covalent bonding between aptamers,mediated for example through ionic bonding, hydrogen bonding,hydrophobic bonding, van der Waals interactions, or a mixture thereof,without any intervening linking moiety joining the individual aptamers.A single multimeric aptamer construct also can use a mixture of covalentbonding, through an intervening linker moiety connecting certainaptamers, and non-covalent bonding, without an intervening linkermoiety, at other bonding sites between aptamers.

The linkers optionally can have one or more functionalities. Forexample, in some embodiments, the linker is sensitive to temperatureand/or pH, meaning that the linker either changes conformation or iscleaved at a pre-designed range of temperature and/or pH.

Any suitable method for making or selecting an aptamer to a target canbe employed to obtain the component aptamers of an aptamer basedmultispecific molecule. For example, aptamers can be identified bySystematic Evolution of Ligands by Exponential Enrichment (SELEX). SELEXis described, for example, in U.S. Pat. No. 5,270,163 which is herebyincorporated by reference. Briefly, SELEX starts with a plurality ofnucleic acids (i.e., candidate aptamer sequences) containing variednucleotide sequences which are contacted with a target. Unbound nucleicacids are separated from those that form aptamer-target complexes. Theaptamer-target complexes are then dissociated, the nucleic acids areamplified, and the steps of binding, separating, dissociating, andamplifying are repeated through as many cycles as desired to yield apopulation of aptamers of progressively higher affinity to the target.Cycles of selection and amplification can be repeated until nosignificant improvement in binding affinity is achieved on furtherrepetitions of the cycle.

The cycles of selection and amplification can be interrupted before asingle aptamer is identified. In such cases, a population of aptamers isidentified, which can offer significant information regarding thesequence, structure, or motifs that allow binding of the aptamer with atarget. Such a population of candidate aptamers also can inform whichportions of the aptamer are not critical for target binding. Thisinformation can then guide the generation of other aptamers to the sametarget. The aptamers thus generated can be used as input for a new roundof SELEX, potentially yielding aptamers with better binding affinitiesor other characteristics of interest.

In some embodiments, candidate aptamer sequences are created thatcontain multimeric aptamer constructs, such as candidate aptamer basedmultispecific molecules, which are then subjected to further rounds ofselection as a multimeric construct. Multimeric candidate aptamerconstructs can be made by linking individual candidate aptamer moietieswith a linking moiety, and optionally using such constructs as input forone or more rounds of SELEX. In some embodiments, individual aptamersare independently selected via one or more rounds of SELEX, and finallylinked together with a linking moiety. Therefore, multimerization ofmonomeric aptamers as well as of multimeric aptamer constructs can beperformed prior to, during, or post SELEX procedures.

The present technology further provides cell redirecting aptamers (e.g.,multivalent aptamers), which can be used as aptameric bridges inaptamer-based CAR immunotherapy systems as well as for in vivo or exvivo genetic modification of cells. The aptameric bridges, cells, kits,and methods of the present technology can be employed in a wide varietyof uses, including as immunotherapies for the treatment of cancers(e.g., hematologic or non-hematologic, individual cells or solidtumors), autoimmune diseases (e.g. arthritis, myasthenia gravis,pemphigus), neuroinflammatory diseases, ophthalmic diseases,neurodegenerative diseases (e.g., ALS, Huntington's disease, Alzheimer'sdisease), neuromuscular diseases (including Duchenne muscular dystrophy,SMA), infectious diseases (e.g., HIV, HSV, HPV, HBV, Ebola,tuberculosis, Cryptococcus), and metabolic diseases (e.g., Type 1diabetes mellitus). They also can be used to provide diagnostic agents,kits, and methods for use in such immunotherapies, including imaging,analysis of cell trafficking, and research and development of newimmunotherapies, as well as to provide prophylaxis when combined withstem cell therapies (e.g., HSCT).

As used herein, “chimeric antigen receptor cells” or “CAR cells” aregenetically modified cells (e.g., T-cells, NK-cells, monocytes, orothers), that have been manipulated ex vivo or in vivo to express asingle-chain variable domain (scFv) antibody fused, through a stalk ortransmembrane domain, to the intracellular domain of a receptor (e.g.,CD3-TCR) so as to endow the cell with the ability to recognize and bindone or more specific antigens and activate a cellular immune response(e.g., kill cancer cells or destroy a virus-infected cell).

As used herein, “antigenic loss” or “antigenic escape” can refer to anyof several mechanisms of resistance or adaptation to immunotherapy, suchas downregulation of a tumor antigen or upregulation of inhibitoryligands (e.g., PD-L1, TIM3, LAG3) which contributes to CAR-T cellfailure, failure of a CAR cell to get to its target (e.g., a tumorsite), immunity against the antibody portion of a CAR (e.g., T-cellresponse against the scFv, particularly if it is not fully humanized),CAR-T cell fitness (i.e., diminished potential for memory self-renewaland increased propensity for exhaustion), or antigen splicing ormutation.

A multimeric aptamer or linked aptamer of the present technologycontains two or more aptamers covalently or non-covalently bound by alinking moiety. According to an embodiment of the technology, the two ormore aptamers can form a CAR-binding portion and a target-bindingportion, each of which contains one or more aptamers. The CAR-bindingaptamer binds to a CAR expressed in an immune cell, such as a T cell,and in some embodiments activates the immune cell but in otherembodiments (e.g., when acting as a “kill” switch) does not activate theimmune cell. The target is an intended target of immunotherapy, i.e., acell intended for elimination. Thus, the CAR-expressing cell andaptameric bridge are intended for use together as a system in animmunotherapy, such as CAR-T cell therapy. Binding of the aptamericbridge to the CAR as well as to the target is preferably high affinitybinding. The target can be a protein (such as a cell-surface receptorprotein), a cell, a small molecule, or a nucleic acid. The target ispreferably located on the surface of a target cell, such as a cancercell, and may or may not be found on other cells (normal cells) of thesubject.

In some embodiments, the target is a tumor antigen, such as CD19, CD20,CD22, CD30, CD123, BCMA, NY-ESO-1, mesothelin, MHC class I-relatedprotein MR1, PSA, PSMA, MART-1, MART-2, Gp100, tyrosinase, p53, ras,Ftt3, NKG2D ligangs, Lewis-Y, MUC1, SAP-1, survivin, CEA, Ep-CAM, Her2,Her3, EGFRvIII, BRCA1/2, CD70, CD73, CD16A, CD40, VEGF-α, VEGF, TGF-β,CD32B, CD79B, cMet, PCSK9, IL-4RA, IL-17, IL-23, 4-1BB, LAG-3, CTLA-4,PD-L1, PD-1, OX-40, or mutated SOD. Component aptamers of an aptamericbridge also can specifically bind to combinations of such targets. Insome embodiments, the target is an antigen of an infectious agent, suchas gag, reverse transcriptase, tat, HIV-1 envelope protein,circumsporozoite protein, HCV nonstructural proteins, hemaglutinins; anaptamer bridge also can specifically bind to combinations of suchtargets.

In a preferred embodiment, the CAR-binding aptamer or aptamers areselected for specific binding to the extracellular domain of a CARhaving affinity for a peptide neo-epitope (PNE), i.e., an anti-PNE CAR.Since the PNE is an epitope that does not exist within the subject'sbody, immune cells expressing the anti-PNE CAR are not activated byendogenous biomolecules, but await the administration to the subject ofthe aptameric bridge, which serves as an “on” switch for the immunecells and targets the CAR-expressing cells toward a desired antigen(s)or cell type(s) bearing the antigen(s). The immune activation and invivo expansion of the CAR-expressing immune cells can be turned off byadministration to the subject of a peptide containing the PNE or ofeither the CAR-binding aptamer or target-binding aptamer of the bridgein monomeric form, any one of which will terminate the activation of theCAR-expressing immune cells by the target.

The PNE can be any peptide epitope not found in the host's proteome(e.g., not found in the human proteome), for which an anti-PNE CAR canbe obtained. An example of a preferred PNE is a peptide fragment of theGCN4 transcription factor from Saccharomyces cerevisiae (NYHLENEVARLKKL,SEQ ID NO:1). A CAR binding GCN4 with high affinity (K_(d)=5.2 μM) andincluding the 52SR4 single chain antibody is described by Rodgers et al.Further PNEs suitable for use with a CAR and corresponding aptamericbridge include: (i) the N-terminal 15-mer peptide ESQPDPKPDELHKSS (SEQID NO:2) of Staphylococcal enterotoxin B, paired with an antibodybinding thereto and described in Clin. Vaccine Immunol. 17(11):1708-1717; (ii) deoxynivalenol, an E. coli mycotoxin, paired with anscFv binding thereto and described at Protein Expr. Purif. 35(1): 84-92;(iii) HPV-16 protein E5, paired with an antibody thereto described atBiomed. Res. Int. 2018; 2018: 5809028; (iv) a rabies virus protein andan scFv binding thereto and described at Protein Expression andPurification 86 (2012) 75-81; (v) an influenza A matrix protein pairedwith an scFv binding thereto and described at Bioconjugate Chem. 2010,21, 1134-1141; (vi) amino acids 134-145 (PRVRGLYFPAGG, SEQ ID NO:3) ofpre-Ω protein of HBV, paired with an scFv binding thereto and describedat Viral Immunol. 2018 May 30; (vii) a VP 3 peptide of duck hepatitisvirus type 1, paired with an scFv described at J. of Virological Methods257 (2018) 73-78; (viii) a peptide (MEESKGYEPP, SEQ ID NO:4) fromGlycoprotein D of bovine herpes virus 1 paired with an scFv described atAppl Microbiol Biotechnol. 2017 December; 101(23-24):8331-8344; (ix) apeptide comprising amino acid 159 of VP1 protein of South AfricanTerritories 2 (SAT2) foot and mouth virus, paired with an scFv bindingthereto and described at Virus Research 167 (2012) 370-379; (x) apeptide (DRTNNQVKA, SEQ ID NO:5) of OmpD from Salmonella typhimurium,paired with an scFv binding thereto and described at VeterinaryMicrobiology 147 (2011) 162-169; (xi) a peptide of isoferritin from E.coli, paired with an scFv binding thereto and described at Journal ofBiotechnology 102 (2003) 177/189; (xii) a peptide (AQEPPRQ, SEQ ID NO:6)located at the N terminus of the grapevine leafroll-associated virus 3coat protein, paired with an scFv binding thereto an described at Arch.Virol. (2008) 153:1075-1084; (xiii) a peptide (PTDSTDNNQNGGRNGARPKQRRPQ,SEQ ID NO:7) of N protein of SARS-CoV, paired with an scFv bindingthereto and described at Acta Biochimica et Biophysica Sinica 2004,36(8): 541-547; (xiv) a peptide containing amino acids 1-15 of HIV Tatprotein, paired with an scFv that binds thereto and is described at J.Virol. 2004 April; 78(7): 3792-3796; and (xv) a peptide from amino acids1363-1454 of the helicase domain of HCV NS3, paired with an scFv bindingthereto and described at J. Hepatology 37 (2002) 660-668, J Virol 1994;68:4829-4836, and Arch Virol 1997; 142:601-610.

Other examples of universal CARs that can be paired with an aptamericbridge of the present technology are described at J. Autoimmun. 2013May. 42:105-16; Blood Cancer J. 2016 August, 6(8): e458; Oncotarget.2017 Dec. 12, 8(65): 108584-108603; Oncotarget 2017 May 9, 8(19):31368-31385; Oncotarget 2018 Jan. 26, 9(7): 7487-7500; and WO2016030414.

A10 RNA aptamer (SEQ ID NO:8) is a 39 nucleotide-long sequence that hasbeen selected against the human prostate-specific membrane antigen(PSMA) and used as a prostate specific delivery agent for siRNA(McNamara et al. 2006-Dassie et al. 2009).

A number of DNA aptamers (SEQ ID NOS:9-110) and RNA aptamers (SEQ IDNOS:111-116) were developed having high affinity binding for human CD3.CELTIC_1s, CELTIC_19s and CELTIC_core are DNA aptamers (SEQ ID NOS: 54,63 and 65), and ARACD3-3700006 and ARACD3-0010209 are RNA aptamers (SEQID NOS:115 and 111), that have all been selected against human CD3.These DNA or 2′-Deoxy-2′-fluoro-thymidine-modified RNA (2′F-RNA)aptamers were purchased from baseclick (Neuried, Germany) as HPLC-RPpurified single stranded oligos synthetized via standard solid phasephosphoramidite chemistry. The anti-CD3 aptamers did not activatecytokine secretion or surface marker expression even when combined withcostimulatory anti-CD28 antibody, and unlike anti-CD3 monoclonalantibodies.

Several consensus sequences for anti-CD3 aptamers were developed.According to these consensus sequences, DNA and RNA aptamers having highaffinity for human CD3 can include the following consensus sequences orvariants thereof:

1. (SEQ ID NO: 117) GX₁X₂TX₃GX₄X₅X₆X₇X₈X₉GGX₁₀CTGG,wherein X₁ is G or A; X₂ and X₆ are A, T, or G; X₃ is T, or G; X₄ andX₉ are G or C; X₅ is C or T; X₇ is T, G, or C; and X₈ and X₁₀are C, T, or A. 2. (SEQ ID NO: 118) GGGX₁TTGGCX₂X₃X₄GGGX₅CTGGC,wherein X₁ and X₂ are A, T, or G; X₃ is T, C, or G; X₄ and X₅are A, T, or C. 3. (SEQ ID NO: 119) GX₁TTX₂GX₃X₄X₅X₆CX₇GGX₈CTGGX₉G,wherein X₁ is A or G; X₂ is T or G;X₃ and X₇, X₉ are G or C; X₄ is T or C;X₅ is A or T; X₆ is T, C, or G; X₈ is A or C. 4. (SEQ ID NO: 120)GGGTTTGGCAX₁CGGGCCTGGC, wherein X₁ is G, C, or T. 5. (SEQ ID NO: 121)GCAGCGAUUCUX₁GUUU, wherein X₁ is U or no base.

EXAMPLES Example 1. Preparation of Bispecific Aptamers Specific for PSMAand CD3

A10 RNA aptamer (SEQ ID NO:8) is a 39 nucleotide-long sequence that hasbeen selected against the human prostate-specific membrane antigen(PSMA) and used as a prostate specific delivery agent for siRNA(McNamara et al. 2006-Dassie et al. 2009).

CELTIC_1s, CELTIC_19s and CELTIC_core are DNA aptamers (SEQ ID NOS: 54,63 and 65), and ARACD3-3700006 and ARACD3-0010209 are RNA aptamers (SEQID NOS:115 and 111), that have all been previously selected againsthuman CD3.

These DNA or 2′-deoxy-2′-fluoro-thymidine-modified RNA (2′F-RNA)aptamers were purchased from baseclick (Neuried, Germany) as HPLC-RPpurified single stranded oligos synthetized via standard solid phasephosphoramidite chemistry. The anti-CD3 aptamers did not activatecytokine secretion or surface marker expression even when combined withcostimulatory anti-CD28 antibody, and unlike anti-CD3 monoclonalantibodies (data not shown).

A10 aptamer was modified with an azide group at its 3′-end forsubsequent triazole inter-nucleotide dimerization. Biotin was added tothe 5′-end of A10 aptamer as a Biotin-TEG that introduces a 16-atommixed polarity spacer between the aptamer sequence and the biotin flag.A Cy5-labelled version of A10 was also synthetized. CELTIC_1s,CELTIC_19s, CELTIC_core, ARACD3-3700006 and ARACD3-0010209 were modifiedwith an alkyne group at their 5′-end for subsequent triazoleinter-nucleotide dimerization. Molecular weight, purity and integritywere verified by HPLC-MS. Affinity and specificity of the A10 anti-PSMARNA aptamer was evaluated on PSMA positive and PSMA negative cells (FIG.3A). Affinity and specificity of anti-CD3 aptamers were evaluated on CD3positive and CD3 negative cells (FIG. 3B).

Anti-PSMA A10 and anti-CD3 aptamers were heterodimerized bycopper-catalyzed click reaction performed for 60 min at 45° C. with theOligo2-Click kit L (baseclick, Neuried, Germany) according tomanufacturers instructions. Reaction products were separated by gelelectrophoresis on 3% agarose gel migrated in 1×TBE buffer (Invitrogen)at 100 V during 30 min. The gels were visualized using Bio-Rad imagingsystem and the results are shown in FIGS. 4A and 4B. Gel slicescorresponding to dimeric aptamers were cut out from the gel and nucleicacids were extracted for 72 h at 8° C. by passive elution in 25 mMNaCl-TE buffer. Bispecific aptamer dimers were recovered by standardsodium acetate precipitation, resuspended in sterile water and stored at−20° C. until use.

Example 2. Functional Stability of Aptamer A10 Specific for PSMA

Stability of A10 RNA aptamer was measured in Dulbecco'sphosphate-buffered saline (DPBS) containing 5% FBS or the FBS alone.Biotinylated aptamer was denatured at 85° C. for 5 min and thenimmediately cooled on ice block to 4° C. for 5 min. The aptamer was thendiluted to a final concentration of 2 μM in DPBS supplemented with 5% ofFBS or in pure FBS. Samples were incubated at 37° C. for 10 min, 30 min,1 h, 2 h, 4 h or 24 h; the control sample contained the freshly preparedaptamers without incubation at 37° C. 100 nM streptavidin-PE was thenadded to each solution and aptamer was incubated with PSMA-positiveLNCaP cells (Human Prostate Carcinoma—ATCC CRL-1740). The half-life ofaptamer A10 in DPBS buffer containing 5% FBS or in pure FBS was thendetermined using flow cytometry on the YL-1 channel, based on thevariation of the fluorescence-positives cells number as a function ofthe incubation time at 37° C. The results of the measurements are shownin FIG. 5 . Aptamer A10 incubated in DPBS buffer containing 5% serum wasstable over 24 h. When tested in pure serum, half of the bindingactivity was lost within the first 2 h of incubation.

Example 3. Determination of the Affinity and Specificity ofAnti-PSMA×Anti-CD3 Bispecific Aptamer to Targets Expressed on Cells

The affinity and specificity of anti-PSMA×anti-CD3 bispecific aptamersto target proteins expressed on cells were evaluated by flow cytometry.These studies were performed on CD3-positive Jurkat (Acute T CellLeukemia Human Cell Line—ATCC TIB-152), CD3-negative Ramos (Burkitt'sLymphoma Human Cell Line—ATCC CRL-1596), PSMA-positive LNCaP (HumanProstate Carcinoma—ATCC CRL-1740) and PSMA-negative PC-3 (Human ProstateCarcinoma—ATCC CRL-1435) cells by incubation with biotinylated RNA/DNAaptamers in SELEX buffer or RNA/RNA aptamers in DPBS buffer,supplemented with 5% of FBS. Cells were cultured in RPMI-1640 medium(Gibco Invitrogen), supplemented with 10% FBS (Gibco Invitrogen) and 1%Penicillin/Steptomycin (Gibco Invitrogen) prior to use. Prior toexperiment, Jurkat, Ramos, LNCaP and PC-3 cells (2.5×10⁵ cells/well)were seeded in 96-well plates and centrifuged at 2500 rpm for 2 min. Thesupernatant was discarded, and the pelleted cells were washed twice with200 μL of SELEX or DPBS-5% FBS buffer preheated at 37° C. Each washingstep was followed by centrifugation at 2500 rpm for 2 min. Aptamers weredenatured at 85° C. for 5 min and immediately placed on ice block of 4°C. for 5 min. Test samples were subsequently diluted at two differentconcentration ranges: 3, 10, 30, 100 and 300 nM (CD3 binding assays) and30, 100 and 300 nM (PSMA binding assays) followed by addition of 100 nMphycoerythrin-labelled streptavidin (streptavidin-PE, eBioscience) toeach solution. Jurkat, Ramos, LNCaP and PC-3 cells were resuspended inthe aptamer dilutions (100 μL/well) and incubated at 37° C. for 30 minin a 5% CO₂ humidified atmosphere. As controls, cells were incubatedwith CD3 monoclonal antibodies (PE-labelled, OKT3 human anti-CD3,Invitrogen), PSMA monoclonal antibodies (Alexa Fluor 488-labelled,GCP-05 human anti-PSMA, Invitrogen), PE-streptavidin, monomeric aptamersor the respective buffers without additional reagents. After incubation,cells were centrifuged at 2500 rpm for 2 min and the supernatant withunbound sequences was discarded. The pelleted cells were washed withSELEX or DPBS-5% FBS buffer (200 μL/well) and centrifuged twice in orderto remove all weakly and non-specifically attached sequences. The cellswere then washed with 1 mg/mL salmon sperm DNA solution (100 μL/well) at37° C. in a 5% CO₂ humidified atmosphere. After 30 min, the salmon spermsolution was removed by centrifugation at 2500 rpm for 2 min and thecells were additionally washed twice with SELEX or DPBS-5% buffer (200μL/well) followed by centrifugation. Jurkat, Ramos, LNCaP and PC-3 cellswith attached DNA or RNA sequences were then fixed (BD CellFIX solution#340181) and the fluorescence-positive cells were counted by flowcytometry (AttuneNXT; Invitrogen, Inc.) on the YL-1 channel.

The results of the binding studies to PSMA-positive cells are shown inFIGS. 6A and 6B. Three RNA/DNA aptamers (A10×CELTIC_1s, A10×CELTIC_19s,A10×CELTIC_core) and two RNA/RNA aptamers (A10×ARACD3-3700006 andA10×ARACD3-0010209) were analyzed along with A10 monomeric aptamer. Forcomparison, binding of the tested reagents to PSMA-negative PC-3 cellswas also measured. A dose-dependent binding to PSMA-positive LNCaP cellswas observed with A10 without reaching saturation of the signal at thehighest tested concentrations. Intensity of the signal was as strong asfor the antibody control. Residual binding of A10 monomer to PC-3 cellswas only observed at the highest tested concentration. All bispecificPSMA×CD3 aptamers exhibited similar binding properties to A10 monomerbut with an improved specificity for target-positive cells as residualbinding to PSMA-negative cells was reduced. For each testedconcentration, signal intensity of bispecific aptamers was superior tothe one measured for A10 monomer, suggesting that heterodimerizationresulted in an improvement of the affinity.

In another experiment, binding of the same aptamers to CD3-positiveJurkat and CD-3 negative Ramos cells was investigated. See FIGS. 7A and7B. As expected A10 aptamer did not bind to these two cell lines.Residual binding of anti-CD3 monomers to Ramos cells was only observedat the highest tested concentration. All bispecific PSMA×CD3 aptamersexhibited similar dose-dependent binding but with superior specificityfor target-positive cells as residual binding to CD3-negative cells wasstrongly reduced. For each tested concentration, signal intensity ofbispecific aptamers was inferior to the one measured for anti-CD3monomers, suggesting that heterodimerization resulted in the lowering ofthe affinity.

Altogether, these results suggest that after heterodimerization thebinding properties of aptamers selected against different targets arenot destroyed due to steric hindrance when evaluated separately.Depending on the selected partners, specificity and affinity forrespective targets may even be improved upon dimerization.

Example 4. Binding of Bispecific Aptamers Targeting PSMA and CD3 asMeasured by Surface Plasmon Resonance

Binding affinity measurements are performed using a BIAcore T200instrument (GE Healthcare). To analyze interactions between aptamers andCD3 and PSMA proteins, 300 Resonance Units of biotinylated aptamers areimmobilized on Series S Sensor chips SA (GE Healthcare) according tomanufacturer's instructions (GE Healthcare). DPBS buffer is used as therunning buffer. The interactions are measured in the “Single KineticsCycle” mode at a flow rate of 30 μl/min and by injecting differentconcentrations of human CD3 ε/γ, CD3 ε/δ, IgG1 Fc and PSMA (AcroBiosystems). The highest aptamer concentration used is 300 nM. Otherconcentrations are obtained by 3-fold dilution. All kinetic data of theinteraction are evaluated using the BIAcore T200 evaluation software.

Comparison of K_(D) values for monomeric and bispecific aptamers areexpected to show that dimerization does not disturb binding propertiesof each subunit for its particular target. Simultaneous binding of PSMAand CD3 ε/γ also can be recorded with the manual injection mode at aflow rate of 10 μl/min and by injecting a solution of the first targetat a saturating concentration followed by a solution of the secondtarget at a saturating concentration. A second injection with aninverted sequence is performed. In both sequences, each injectionresulting in responses of equal intensities indicates that both arms ofbispecific aptamers are able to bind the second target while the bindingsite for the first antigen is occupied. Monomers failing to respond toboth injections of target solutions indicate that the bispecific aptamercan simultaneously bind both targets.

Example 5. Bioactivity of Bispecific Aptamers Specific for PSMA and CD3

Cytotoxicity assays were carried out on unstimulated peripheral bloodmononuclear cells (PBMCs). Freshly prepared PBMCs were isolated frombuffy coats obtained from healthy donors (Etablissement Français duSang, Division Rhônes-Alpes). After diluting the blood with DPBS, thePBMCs were separated over a FICOLL density gradient (FICOLL-PAQUEPREMIUM 1.077 GE Healthcare), washed twice with DPBS, resuspended inRPMI-1640 medium (Gibco Invitrogen) to obtain a cell density of 5×10⁶cells/ml. These PBMCs were used as effector cells.

LNCaP target cells were labeled with 2 μM calcein AM (Trevigen Inc,Gaithersburg, Md., USA) for 30 min at 37° C. in cell culture medium. Thecalcein AM fluorochrome is a dye that is trapped inside live LNCaP cellsand only released upon redirected lysis. After 2 washes in cell culturemedium, a cell density of 5×10⁵ cells/ml was adjusted in RPMI-1640medium and 100 μl aliquots of 50,000 cells were used per assay reaction.A standard reaction at 37° C./5% CO₂ lasted for 4 hr and used 5×10⁴cells calcein AM-labeled target cells, 5×10⁵ PBMCs (E/T ratio of 1:10)and 20 μl of bispecific aptamer solutions at 1 μM in a total volume of200 μl. After the cytotoxic reaction, the released dye in the incubationmedium was quantitated in a fluorescence reader (VarioSkan Lux,ThermoFisher, Waltham, Mass., USA) and compared with the fluorescencesignal from a control reaction in which the cytotoxic compound wasabsent and a reaction in which the fluorescence signal was determinedfor totally lysed cells (where aptamers were replaced by A100 reagentpurchased from Chemometec, Allerod, Denmark). On the basis of thesereadings, the specific cytotoxicity was calculated according to thefollowing formula: [fluorescence (sample)−fluorescence(control)]/[fluorescence (total lysis)−fluorescence (control)]×100.

The results of the cytotoxicity assay obtained after 4 h incubation inpresence of aptamers 100 nM with a single E:T ratio of 10:1 are shown inFIG. 8 . Null to weak specific cell killing activity (<10%) was observedwith PSMA×CD3 bispecific RNA/DNA aptamers. Superior specificcytotoxicity was measured with RNA/RNA aptamers A10×ARACD3-3700006 andA10×ARACD3-0010209 that induced the killing of 40-50% of LNCaP cells.Control monomer A10 lacking the CD3 binding moiety did not induce anycytotoxicity.

These results suggest that engineered aptamer switches are able torecruit effector T lymphocytes to target cells to redirect theircytolytic machinery and eliminate a particular cell population.

Example 6. Treatment of Cancer in a Preclinical Model withAnti-CD3×Anti-PSMA Aptamer

In vivo efficacy and toxicity of different multimeric aptamer constructsin comparison to monomeric aptamers in mice are evaluated. Adult micebearing PSMA positive tumors are administered with aptamers thatspecifically bind to CD3 and PSMA, in different groups of mice, theaptamers are either in monomeric form or multimeric form. Efficacy isevaluated by measuring tumor size, tumor growth and rate, and survivalin the treated groups versus controls. Toxicity is assessed by theincidence of adverse reactions in treated groups versus controls.

Example 7. Treatment of Cancer in a Preclinical Model withAnti-CD3×Anti-PSMA Aptamer

In vivo efficacy and toxicity of different multimeric aptamer constructsin comparison to monomeric aptamers in mice are evaluated. Adult micebearing PSMA positive tumors are administered aptamers that specificallybind to CD3 and PSMA, in different groups of mice, the aptamers areeither in monomeric form or multimeric form. Efficacy is evaluated bymeasuring tumor size, tumor growth and rate, and survival in the treatedgroups versus controls. Toxicity is assessed by the incidence of adversereactions in treated groups versus controls.

Example 8. Preparation of Bispecific Aptamers Specific for PSMA andCAR-PN E

ARAA-00100001 and ARAA-01700001 aptamers were purchased from baseclick(Neuried, Germany) as HPLC-RP purified 2′-F RNA oligos synthetized viastandard solid phase phosphoramidite chemistry.

A10 2′F-RNA aptamer was modified with an azide group at its 3′-end forsubsequent triazole inter-nucleotide dimerization. Biotin was added tothe 5′-end of A10 aptamer as a Biotin-TEG that introduces a 16-atommixed polarity spacer between the aptamer sequence and the biotin flag.ARAA-00100001 and ARAA-01700001 were modified with an alkyne group attheir 5′-end for subsequent triazole inter-nucleotide dimerization.Molecular weight, purity and integrity were verified by H PLC-MS.

The procedure described in Example 1 was used to prepare bispecificanti-PSMA A10 and anti-CAR PNE aptamers. The gels were visualized usingBio-Rad imaging system and the results are shown in FIG. 4A. Gel slicescorresponding to dimeric aptamers were cut out from the gel and nucleicacids were extracted for 72 h at 8° C. by passive elution in 25 mMNaCl-TE buffer. Bispecific aptamer dimers were recovered by standardsodium acetate precipitation, resuspended in sterile water and stored at−20° C. until use.

Example 9. Determination of the Affinity and Specificity ofAnti-PSMA×Anti-CAR PNE Bispecific Aptamer to Targets Expressed on Cells

The affinity and specificity of anti-PSMA×anti-CAR PNE aptamers totarget proteins expressed on cells were evaluated by flow cytometry.These studies were performed on PSMA-positive LNCaP (Human ProstateCarcinoma—ATCC CRL-1740) and PSMA-negative PC-3 (Human ProstateCarcinoma—ATCC CRL-1435) in DPBS buffer containing 5% FBS, as describedin Example 3. Aptamers were tested within a single concentration range:30, 100 and 300 nM.

The results of the binding studies to PSMA-positive cells are shown inFIG. 6A. Two RNA/RNA aptamers, A10×ARAA-00100001 and A10×ARAA-01700001were analyzed along with A10 monomeric aptamer. For comparison, bindingof the tested reagents to PSMA-negative PC-3 cells was also measured.

A dose-dependent binding to PSMA-positive LNCaP cells was observed withA10 without reaching saturation of the signal at the highest testedconcentrations. Intensity of the signal was as strong as for theantibody control. Residual binding of A10 monomer to PC-3 cells was onlyobserved at the highest tested concentration. Both bispecific PSMA×CARPNE aptamers exhibited similar binding properties to A10 monomer butwith an improved specificity for target-positive cells as residualbinding to PSMA-negative cells was reduced. For each testedconcentration, signal intensity of bispecific aptamers was superior tothe one measured for A10 monomer, suggesting that heterodimerizationresulted in an improvement of the affinity.

Altogether the results from Example 9 and this example suggest that theheterodimerization of aptamers selected against different targets doesnot significantly impact the binding properties of each moiety whenevaluated separately.

Example 10. Bioactivity of Bispecific Aptamers Specific for CAR-PNE andPSMA

Cytotoxicity assays are carried out on unstimulated peripheral bloodmononuclear cells (PBMCs). Freshly prepared PBMCs are isolated frombuffy coats obtained from healthy donors (Etablissement Francais duSang, Division Rhones-Alpes). After diluting the blood with DPBS, thePBMCs are separated over a FICOLL density gradient (FICOLL-PAQUE PREMIUM1.077 GE Healthcare), washed twice with DPBS, resuspended in RPMI-1640medium (Gibco Invitrogen) to obtain a cell density of 5×10⁶ cells/mi.These PBMCs are transduced with lentiviral vectors expressing theCAR-PNE receptor. These PBMC-CAR-PNE are used as effector cells.

LNCaP target cells are labeled with 2 μM calcein AM (Trevigen Inc,Gaithersburg, Md., USA) for 30 min at 37° C. in cell culture medium. Thecalcein AM fluorochrome is a dye that is trapped inside live LNCaP cellsand only released upon redirected lysis. After 2 washes in cell culturemedium, a cell density of 5×10⁵ cells/mi is adjusted in RPMI-1640 mediumand 100 μl aliquots of 50,000 cells are used per assay reaction. Astandard reaction at 37° C./5% CO₂ lasts for 4 hr and uses 5×10⁴ cellscalcein AM-labeled target cells, 5×10⁵ PBMCs-CAR-PNE (ETT ratio of 1:10)and 20 μl of bispecific aptamer solutions at 1 μM in a total volume of200 μl. After the cytotoxic reaction, the released dye in the incubationmedium is quantitated in a fluorescence reader (VarioSkan Lux,ThermoFisher, Waltham, Mass., USA) and compared with the fluorescencesignal from a control reaction in which the cytotoxic compound is absentand a reaction in which the fluorescence signal is determined fortotally lysed cells (where aptamers were replaced by A100 reagentpurchased from Chemometec, Allerod, Denmark). On the basis of thesereadings, the specific cytotoxicity is calculated according to thefollowing formula: [fluorescence (sample)−fluorescence(control)]/[fluorescence (total lysis)−fluorescence (control)]×100. Theresults of the cytotoxicity assay are obtained after 4 h incubation inpresence of aptamers 100 nM with a single E:T ratio of 10:1. Specificcytotoxicity is measured with RNA/RNA aptamers A10×CAR PNE that inducedthe killing of more than 30% of LNCaP cells. Control monomer A10 lackingthe CAR PNE binding moiety is also checked for cytotoxicity.

The engineered aptamer switches should be able to recruit effector Tlymphocytes to target cells to redirect their cytolytic machinery andeliminate a particular cell population.

Example 11. Treatment of Cancer in a Preclinical Model with a CAR-TAptameric Switch

In vivo efficacy and toxicity of switch aptamer constructs in comparisonto monomeric aptamers in mice are evaluated. Multimeric aptamers areprepared as switches that will turn on the activity of CAR T-cell basedtherapeutics. Adult mice bearing tumors are first injected with T cellstransduced with CAR PNE and the multimeric aptamer made of an anti-CARPNE aptamer fused to PSMA, or CD19, or CD2 or CD22 tumor associatedtargets is infused. Efficacy is evaluated by measuring tumor size, tumorgrowth and rate, and survival in the treated groups versus controls.Toxicity is assessed by the incidence of adverse reactions in treatedgroups versus controls.

TABLE 1 Summary of Sequences Sequence SEQ ID Description Sequence NO:GCN4 NYHLENEVARLKKL 1 Transcription Factor (PNE) N-terminalESQPDPKPDELHKSS 2 peptide of Staph, entertoxin B (PNE) HBV pre-S2PRVRGLYFPAGG 3 protein (aa 134- 145) (PNE) Bovine Herpes MEESKGYEPP 4Virus Glycoprotein D (PNE) Salmonella DRTNNQVKA 5 typhimuriumOmp D peptide (PNE) Grapevine AQEPPRQ 6 leafroll- associated virus3 coat protein N- terminal peptide (PNE) SARS-CoV-1 NPTDSTDNNQNGGRNGARPKQRRPQ 7 protein peptide A10 RNAGGGAGGACGAUGCGGAUCAGCCAUGUUUACGUCACUCCU 8 aptamer anti- PSMA Cluster 1CCGGGTGGGGGTTTGGCACCGGGCCTGGCGCAGGGATTCG 9 Cluster 2GAGGGGTTTGGCATCGGGCCTGGCGCCATTCAAGCTATGC 10 Cluster 3GCGTAAGGGTTTGGCAGCGGGCCTGGCGGAACGCGTGTAT 11 Cluster 4GGAGTGGAGTATTCCGGGTTTGGCATCGGGCCTGGCGAAG 12 Cluster 5CGGCAGGGGTTTGGCTCCGGGTCTGGCGAACTGGCTGAGA 13 Cluster 6AAGGGATTGGCGTCGGGCCTGGCGTAAGGAGGCTATGCTC 14 Cluster 7GGGATTGGCGCTGGGCCTGGCAAGGAATCTTCTCGTTGTA 15 Cluster 8GGGATTGGCTTCGGGCCTGGCGAGTATTGTTTTCCTGGAG 16 Cluster 9GCATCGAAATGGGGTTGGCACCGGGCCTGGCGAATTGGAT 17 Cluster 10GAGACTAGAGGGATTGGCTTCGGGCCTGGCGTAC 18 Cluster 11GATGGAGGGTTTGGCGGTGGGCCTGGCAAGTTATCTCATA 19 Cluster 12TACGGCTAGGGTTTGGCGTTGGGCCTGGCAGGACCGTAAG 20 Cluster 13ATATGGGAGGGTGAGGGTTTGGCTGCGGGCCTGGCGGGAG 21 Cluster 14TGCGGCACATGTACGCGGAGGGATTGGCATAGGGTCTGGC 22 Cluster 15GGGGTTGGCTTTGGGCCTGGCAGTCATTTGTGAATCCTTA 23 Cluster 16TCCGACAAAAGGGATTGGCTTCGGGCCTGGCGGGGTTGCC 24 Cluster 17GGTCGGGGTTTGGCATCGGGACTGGCGTTATACAATCGT 25 Cluster 18GATGGGGTTTGGCGTCGGGCCTGGCGAATACATCTAAAAG 26 Cluster 19TACCGCGGGGATTGGCTCCGGGCCTGGCGTCGTAATCTGA 27 Cluster 20GGGGTTTGGCTGCGGGCCTGGCGCATGATTCAACGAGACA 28 Cluster 21GGTCGGGTGCTACTGAGCGATTGGCTTTCCGGACTGGGGA 29 Cluster 22CGACCACAGGGGTTTGGCTTCGGGACTGGCGGTGGGCACT 30 Cluster 23CGACCACAGGGGTTTGGCTTCGGGACTGGCGGTGGGCACT 31 Cluster 24TATGGGTTTGGCATCGGGCCTGGCGGAATGGAAAATGTTA 32 Cluster 25AGACGGGTTTGGCTGCGGGCCTGGCGGTCGTCATTCCTCT 33 Cluster 26GAGGGGATTGGCATTTGGGCCTGGCAAATTCATCTATTCT 34 Cluster 27AGGGGTTTGGCGTCGGGCCTGGCGCAGCTCTTCTTGTGTTT 35 Cluster 28GGGATTGGCTTCGGGCCTGGCGTATCTTTTACATTACC 36 Cluster 29GGTGGACGGTATACAGGGGCTGCTCAGGATTGCGGATGAT 37 Cluster 30CCGTTTGAAGCGTTAGGGTTTGGCATCGGGCCTGGCGCAC 38 Cluster 31AGGGTTTGGCTACGGGCCTGGCGAGCTGTTTCCGCTACTC 39 Cluster 32GTGTTATGATACTATGCGTATGGATTGCAAAGGGCTGCTG 40 Cluster 33GAAGGGTTTGGCATTGGGCCTGGCAAGATAATTTGCAAGT 41 Cluster 34CGGCGAAGTGGCAGGGTTTGGCTTCGGGTCTGGCGGAACA 42 Cluster 35GAGGGTTTGGCAGTGGGCCTGGCATCAATTCTTTGTTTTC 43 Cluster 36TACTGAGGGTTTGGCATTGGGCCTGGCATATTGGTATTT 44 Cluster 37ATGGGTTTGGCACCGGGTCTGGCGGATTCGATAGGTGGTT 45 Cluster 38GGGGGTTTGGCTCTGGGCCTGGCATAACGAACCTTCGGAG 46 Cluster 39TGCCCGAGAGGACTGCTTAGGCTTGCGAGTAGGGAACGCT 47 Cluster 40AGTGGGATTGGCTTCGGGCCTGGCGTTCGCAACATGTTTA 48 Cluster 41GGGGATTGGCACTGGGACTGGCACCTTTTTAACATGTATG 49 Cluster 42GCAATTAAGGGATTGGCTCCGGGCCTGGCGCCACGCATGG 50 Cluster 43TGGGGTTTGGCAGCGGGTCTGGCGATCATAATGGTGTGCG 51 Cluster 44ACGGGGGATTGGCTTTGGGCCTGGCAATTAATTTACTGTT 52 Cluster 45GAGCGCTTGGCAGCCGGTCTGGGGACATCAGAGGTGATGG 53 CELTIC_1sTTTCCGGGTGGGGGTTTGGCACCGGGCCTGGCGCAGGGAT 54 TCG CELTIC_2sGAGGGGTTTGGCATCGGGCCTGGCGCCATTCAAGCTATGC 55 CELTIC 3sGCGTAAGGGTTTGGCAGCGGGCCTGGCGGAACGCGTGTAT 56 CELTIC 21sGGTCGGGTGCTACTGAGCGATTGGCTTTCCGGACTGGGGA 57 CELTIC 4sGGAGTGGAGTATTCCGGGTTTGGCATCGGGCCTGGCGAAG 58 CELTIC 5sCGGCAGGGGTTTGGCTCCGGGTCTGGCGAACTGGCTGAGA 59 CELTIC 6sAAGGGATTGGCGTCGGGCCTGGCGTAAGGAGGCTATGCTC 60 CELTIC_9sGCATCGAAATGGGGTTGGCACCGGGCCTGGCGAATTGGAT 61 CELTIC 11sGATGGAGGGTTTGGCGGTGGGCCTGGCAAGTTATCTCATA 62 CELTIC 19sTACCGCGGGGATTGGCTCCGGGCCTGGCGTCGTAATCTGA 63 CELTIC 22sCGACCACAGGGGTTTGGCTTCGGGACTGGCGGTGGGCACT 64 CELTIC coreGGGXTTGGCXXXGGGXCTGGC 65 CELTIC core_1 GGGTTTGGCACCGGGCCTGGCGC 66CELTIC core_2 GGGTTTGGCACCGGGCCTGGC 67 CELTIC core_3 CCGGGCCTGGCC 68CELTIC core_4 GGGTTTGGCATCGGGCCTGGCG 69 CELTIC core_5GGGTTTGGCGGTGGGCCTGGC 70 CELTIC core_6 TTTGGGTTTGGCACCGGGCCTGGC 71CELTIC core_T TTTGGGTTTGGCATCGGGCCTGGC 72 CELTIC core_7GGGTTTGCACCGGGCCTGGC 73 CELTIC core_8 GGGTTTGCACCGGGCCTGGC 74CELTIC core_9 GGGTTTGGACCGGGCCTGGC 75 CELTIC GGGTTTGGCACC_GGCCTGGC 76core_10 CELTIC GGGTTTGGCACCGG_CCTGGC 77 core_11 CELTICGGGTTTGGCACCGGG_CTGGC 78 core_12 CELTIC GGGTTTGGCACCGGGC_TGGC 79 core_13CELTIC _GGTTTGGCATCGGGCCTGGC 80 core_14 CELTIC G_GTTTGGCATCGGGCCTGGC 81core_15 CELTIC GG_TTTGGCATCGGGCCTGGC 82 core_16 CELTICGGG_TTGGCATCGGGCCTGGC 83 core_17 CELTIC GGGT_TGGCATCGGGCCTGGC 84 core_18CELTIC GGGTT_GGCATCGGGCCTGGC 85 core_19 CELTIC GGGTTT_GCATCGGGCCTGGC 86core_20 CELTIC GGGTTTG_CATCGGGCCTGGC 87 core_21 CELTICGGGTTTGG_ATCGGGCCTGGC 88 core_22 CELTIC GGGTTTGGC_TCGGGCCTGGC 89 core_23CELTIC GGGTTTGGCA_CGGGCCTGGC 90 core_24 CELTIC GGGTTTGGCAT_GGGCCTGGC 91core_25 CELTIC GGGTTTGGCATC_GGCCTGGC 92 core_26 CELTICGGGTTTGGCATCG_GCCTGGC 93 core_27 CELTIC GGGTTTGGCATCGG_CCTGGC 94 core_28CELTIC GGGTTTGGCATCGGG_CTGGC 95 core_29 CELTIC GGGTTTGGCATCGGGC_TGGC 96core_30 CELTIC GGGTTTGGCATCGGGCC_GGC 97 core_31 CELTICGGGTTTGGCATCGGGCCT_GC 98 core_32 CELTIC GGGTTTGGCATCGGGCCTG_C 99 core_33CELTIC GGGTTTGGCATCGGGCCTGG_ 100 core_34 CELTIC GGGTTTGGGATCGGGCCTGGC101 core_35 CELTIC GGGTTTGGCATCGGGCCTGGG 102 core_36 CELTICGGGTTTGGGATCGGGCCTGGC 103 core_37 CELTIC GGGTTTGGCATCGGGACTGGC 104core_38 CELTIC GGGTTTGGCATCGGGGCTGGC 105 core_39 CELTICGGGTTTGGCATCGGGTCTGGC 106 core_40 CELTIC GGGTTTGGCATCGGGCTGGC 107core_41 CELTIC GGGTTTGGCA_CGGG_CTGGC 108 core_42 CELTICGGGTTTGGCAGCGGG_CTGGC 109 core_43 CELTIC GGGTTTGGCAACGGG_CTGGC 110core_44 ARACD3- UCUAAGCAAUAUUGUUUGCUUUUGCAGCGAUUCUGUUUCGAU 111 0010209AUAUUA ARACD3- UUCAAGAUAAUGUAAUUAUUUUUGCAGCGAUUCUUGUUUUGU 112 2980001UCGAUUU ARACD3- CAAAGUUCAAGAUUGAGCUUUUUGCAGCGAUUCUUGUUUUAU 113 0270039CAAACGA ARACD3- GAUGAUAUCUUUAAUAUCAAUUGCAGCGAUUCUUGUUUGAGA 114 3130001AUAAAC ARACD3- UAUAGACUUUAAUGUCUCAUUUUCGCAGCGAUUCUUGUUUAU 115 3700006UUAACAUA Core_sequence UXGCAGCGAUUCUXXUU 116 RNA Consensus-1GX₁X₂TX₃GX₄X₅X₆X₇X₈X₉GGX₁₀CTGG, 117 wherein X₁ is G or A; X₂ andX₆ are A, T, or G; X₃ is T, or G; X₄ and X₉ are G or C; X₅ is C or T; X₇is T, G, or C; and X₈ and X₁₀ are C, T, or A. Consensus-2GGGX₁TTGGCX₂X₃X₄GGGX₅CTGGC, wherein X₁ and X₂ are A, T, 118or G; X₃ is T, C, or G; X₄ and Xs are A, T, or C. Consensus-3GX₁TTX₂GX₃X₄X₅X₆CX₇GGX₈CTGGX₉G, 119 wherein X is A or G; X₂ isT or G; Xs and X₇, X₉ are G orC; X₄ is T or C; X₅ is A or T; X₆ is T, C, or G; X₈ is A or C.Consensus-4 GGGTTTGGCAX₁CGGGCCTGGC, wherein X₁ is G, C, or T. 120Consensus-5 GCAGCGAUUCUX₁GUUU, wherein X₁ is U or nothing 121DNA aptamer TAGGGAAGAGAAGGACATATGAT-(N40)- 122 libraryTTGACTAGTACATGACCACTTGA forward primer TAGGGAAGAGAAGGACATATGAT 123for DNA SELEX reverse primer TCAAGTGGTCATGTACTAGTCAA 124 for DNA SELEXRNA aptamer CCTCTCTATGGGCAGTCGGTGAT-(N20)- 125 libraryTTTCTGCAGCGATTCTTGTTT-(N10)- GGAGAATGAGGAACCCAGTGCAG forward primerTAATACGACTCACTATAGGGCCTCTCTAT 126 for RNA SELEX GGGCAGTCGGTGATreverse primer CTGCACTGGGTTCCTCATTCTCC 127 for RNA SELEXforward blocking ATCACCGACTGCCCATAGAGAGG 128 sequence for RNA SELEXreverse blocking CTGCACTGGGTTCCTCATTCTCC 129 sequence for RNA SELEXcapture CAAGAATCGCTGCAG 130 sequence for RNA SELEX

As used herein, “consisting essentially of” allows the inclusion ofmaterials or steps that do not materially affect the basic and novelcharacteristics of the claim. Any recitation herein of the term“comprising”, particularly in a description of components of acomposition or in a description of elements of a device, can beexchanged with “consisting essentially of” or “consisting of”.

While the present invention has been described in conjunction withcertain preferred embodiments, one of ordinary skill, after reading theforegoing specification, will be able to effect various changes,substitutions of equivalents, and other alterations to the compositionsand methods set forth herein.

What is claimed is:
 1. An aptamer-based multispecific antigen bindingmolecule comprising 1) two or more target binding aptamer regions havingbinding specificities for different targets, and 2) one or more linkersconnecting the aptamer regions, wherein the linker comprises a clickchemistry product.
 2. The aptamer-based multispecific antigen bindingmolecule of claim 1, wherein the linker further comprises a linkermoiety selected from the group consisting of a covalent bond, asingle-stranded nucleic acid, a double-stranded nucleic acid,self-assembling complementary oligonucleotides, a peptide, apolypeptide, an oligosaccharide, a polysaccharide, a synthetic polymer,a hydrazone, a thioether, an ester, a triazole, a nanoparticle, amicelle, a liposome, a cell, and combinations thereof.
 3. Theaptamer-based multispecific antigen binding molecule of any of thepreceding claims that can bind to specific targets on one or more ofhuman cells, immune cells, cancer cells, genetically modified cells,bacteria, or viruses.
 4. The aptamer-based multispecific antigen bindingmolecule of any of the preceding claims that can redirect the binding ofone cell type from one target cell to another target cell.
 5. Theaptamer-based multispecific antigen binding molecule of any of thepreceding claims that can form a bridge between an immune cell and acancer cell.
 6. The aptamer-based multispecific antigen binding moleculeof any of the preceding claims that can stimulate and activate an immunecell.
 7. The aptamer-based multispecific antigen binding molecule ofclaim 6, wherein the immune cell is a T-cell, NK-cell, or macrophage,and said binding leads to destruction of a target cell bound to a targetbinding aptamer of the aptamer based multispecific antigen bindingmolecule.
 8. The aptamer-based multispecific antigen binding molecule ofany of the preceding claims, wherein the molecule possesses a bindingspecificity for an antigen selected from the group consisting of CD3,CD8, CD4, CD19, Epithelial cell adhesion molecule, CD20, CD22, CD123,BCMA, B7H3, CEA, PSMA, Her2, CD33, CD38, DLL3, EGF-R, NKG2D ligands, MHCclass I-related protein MR1, mesothelin, PD-1, PD-L1, CTLA04, Lag-3,TIM-3, OX40, CD47, VEGF, PRAME, NY-ESO-1, MAGE A4, MAGE A3/A6, MAGE A10,and AFP.
 9. The aptamer-based multispecific antigen binding molecule ofclaim 3, wherein the molecule binds to an immune cell expressing CD3antigen.
 10. The aptamer-based multispecific antigen binding molecule ofclaim 1, wherein the molecule binds PSMA antigen on a cancer cell. 11.The aptamer-based multispecific antigen binding molecule of claim 1comprising one or more CD3 antigen binding region that can bind to aT-cell and one or more PSMA antigen binding region that can bind to aPSMA expressing cell, wherein the CD3 antigen binding region and thePMSA antigen binding region are connected by one or more linkers. 12.Use of the aptamer-based multispecific antigen binding molecule of claim11 in the treatment of a PSMA expressing cancer including prostatecancer.